Immunoassay of serum alpha(1)-antitrypsin by affinity-probe capillary isoelectric focusing using a fluorescence-labeled recombinant antibody fragment

An immunoassay for human alpha(1)-antitrypsin based on affinity-probe capillary isoelectric focusing (AP-CIEF) is described. The method is based on the separation of free and bound labeled antibody fragments by CIEF with laser-induced fluorescence detection. A recombinant Fab' fragment of mouse...

Full description

Saved in:
Bibliographic Details
Published in:Electrophoresis 2002-03, Vol.23 (6), p.909-917
Main Authors: Shimura, Kiyohito, Hoshino, Makoto, Kamiya, Kei-ichiro, Katoh, Kohgoro, Hisada, Sunao, Matsumoto, Hiroyuki, Kasai, Ken-ichi
Format: Article
Language:English
Subjects:
Acrylamides
alpha 1-Antitrypsin - analysis
alpha 1-Antitrypsin - immunology
Animals
Electrophoresis, Capillary - methods
Fluorescence
Fluorescent Dyes
Humans
Immunoassay - methods
Immunoglobulin Fab Fragments - genetics
Immunoglobulin Fab Fragments - immunology
Immunoglobulin G - genetics
Immunoglobulin G - immunology
Mice
Recombinant Fusion Proteins - genetics
Recombinant Fusion Proteins - immunology
Rhodamines
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:An immunoassay for human alpha(1)-antitrypsin based on affinity-probe capillary isoelectric focusing (AP-CIEF) is described. The method is based on the separation of free and bound labeled antibody fragments by CIEF with laser-induced fluorescence detection. A recombinant Fab' fragment of mouse immunoglobulin G1 (IgG1) against human alpha(1)-antitrypsin was labeled with tetramethylrhodamine on the single cysteine residue at the hinge region. A single pI isomer of the labeled Fab' was purified by IEF in a slab of agarose gel and was then used as the affinity probe for alpha(1)-antitrypsin. The use of recombinant Fab' considerably simplified the labeling process. Although there was some difficulty in the quantification of native alpha(1)-antitrypsin with the affinity probe, carbamylation of the antigen sample by heat treatment with urea made the complex peaks appear reproducibly and more distinct, thus facilitating the identification and quantification of the complex. The system provided an almost linear response to a pure sample of alpha(1)-antitrypsin over a concentration range of 5-1000 ng/mL and the detection limit extended down to around 2 ng/mL. Alpha(1)-antitrypsin in a serum sample was determined using this system to be 1.2 mg/mL which is comparable to the reported value for the protein.
ISSN:0173-0835
1522-2683
DOI:10.1002/1522-2683(200203)23:6<909::AID-ELPS909>3.0.CO;2-F